Sulfated alginate oligosaccharide exerts antitumor activity and autophagy induction by inactivating MEK1/ERK/mTOR signaling in a KSR1-dependent manner in osteosarcoma

Alginate oligosaccharide (AOS) has the function to inhibit tumor progression and the sulfated modification can enhance the antitumor activity. To date, the function and mechanism of sulfated AOS (AOS-SO4) in tumors remain largely elusive. We prepared AOS by the enzymatic degradation of alginate, collected AOS-SO4 by sulfating following the canonical procedure. Using these materials, in vitro assays showed that both AOS and AOS-SO4 elicited antitumor effects in osteosarcoma cells. Sulfated modification significantly enhanced the antitumor activity. In addition, AOS-SO4 had obvious effects on cell cycle arrest, apoptosis, and autophagy induction in vitro and in vivo. Mechanistically, we observed that AOS-SO4 treatment triggered proapoptotic autophagy by inhibiting MEK1/ERK/mTOR signaling. The ERK activator reversed AOS-SO4-induced autophagy. More importantly, we found that KSR1 interacted with MEK1 and functioned as a positive regulator of MEK1 protein in osteosarcoma cells. High KSR1 expression was significantly associated with poor survival in osteosarcoma patients. Together, these results suggest that AOS-SO4 has a better antitumor effect in osteosarcoma by inhibiting MEK1/ERK/mTOR signaling, which is KSR1-dependent; thus, AOS-SO4 can be a new potential therapeutic candidate for the treatment of osteosarcoma.

clones were fixed with 4% paraformaldehyde and dyed with 0.1% crystal violet. Finally, the cell colonies were counted.
Cell migration and invasion assays. Cell migration and invasion ability were assessed through transwell filter chambers (Corning, New York, NY, USA), which were 8-mm pore size chamber inserts in a 24-well plate. For the migration assay, 48 h after the IC50 of AOS and AOS-SO4 treatment, 200 µL of serum-free medium containing 5×10 4 MNNG and U2OS cells or 1×10 5 MG63 cells was dropped into the upper chambers. For the invasion assay, 1×10 5 MNNG, U2OS cells or 2×10 5 MG63 cells were placed into the upper chambers, which were coated with Matrigel diluted with serum-free culture medium. Separately, 800 µL of culture medium supplemented with 10% FBS was added to the lower chambers. After incubation at 37°C, the cells on the bottom surface of the membrane were stained and counted.
Wound-healing assay. Osteosarcoma cells were seeded in 6-well plates to reach 90% confluence.
Then, the wounds were scraped/wounded using a yellow tip, treated with different drugs (the IC50 of AOS and AOS-SO4) and measured at 0 h, 12 h, and 24 h by microscopy.
Western blot analysis. The tumor tissue was weighed and added to RIPA lysis buffer (Thermo Fisher, Waltham, USA) at a ratio of 1:8 (1 g tissue plus 8 ml RIPA lysis buffer). Protease and phosphatase inhibitors were added at a ratio of 100:1. The tissue was homogenized with a homogenizer on ice, mixed and sucked into an EP tube. In addition, cells were scraped into EP tubes with cell scraping tools and mixed with RIPA lysis buffer. All operations were carried out on ice. After measuring the protein concentration with BCA protein assays (Thermo Fisher, Waltham, USA), 5× SDS loading buffer was added to each EP tube and heated at 95°C for 5 min. Proteins were separated by 8%-12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). The protein mass marker was a mixture of proteins from 10 kDa to 245 kDa (Yeasen, Shanghai, China, 20352ES76). The membranes were incubated with 1:1000-5000 primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. Finally, an ECL detection system (SmartChemi 420, Beijing, China) was used to measure the immune reaction zone.
Western blot images were quantified using ImageJ software. First-strand cDNA was synthesized with a PrimeScript RT Reagent Kit (Takara, Dalian, China). The cDNA templates were combined with SYBR Green premix with Rox II (Takara, Dalian, China) to perform quantitative real-time polymerase chain reaction (qRT-PCR). Information on the primer sequences used was presented in Supplementary Table 2.

Coimmunoprecipitation. Coimmunoprecipitation was performed in osteosarcoma cells. Equal
amounts of protein (3000 μg) were incubated with antibodies at 4 ℃ overnight followed by incubation with protein A/G magnetic beads (Biotool) for 3 h at 4 ℃. The beads were washed using phosphate-buffered saline (PBS) containing 1% Triton X-100. IgG-bound, HA-bound or MEK1-bound proteins were separated using SDS-PAGE for Western blot analysis.

Confocal immunofluorescence. Confocal immunofluorescence was performed on osteosarcoma cells.
Cells were fixed and incubated with a mouse monoclonal anti-HA antibody and a rabbit polyclonal anti-MEK1 antibody overnight at 4 ℃ followed by 1 h incubation with secondary antibodies (Invitrogen). The cells were incubated with a 1:1000 dilution of 4ʹ,6-diamidino-2-phenylindole (DAPI) for 5 min and viewed with a Fluoview FV1000 microscope (Olympus).
Transmission electronic microscopy. Cells treated with or without AOS-SO4 were harvested, fixed, dehydrated, embedded in Epon, stained and observed. Images were acquired using an HT7700 electron microscope (Hitachi, Tokyo, Japan) to observe the autophagic vacuoles. The number of autophagic vacuoles (AVs) in each cell was quantified using 20 randomly selected cells of each group.
Clinical samples and IHC. The tissue microarray contained 100 patients diagnosed with osteosarcoma at Shanghai Sixth People's Hospital. They received primary surgical treatment and preoperative and postoperative neoadjuvant therapy. Ethics approval was obtained from the Ethics Committee of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital, and written informed consent was obtained from each patient prior to sample collection. A statement confirmed that all methods were carried out in accordance with relevant guidelines and regulations. A standard IHC staining procedure was followed. Briefly, paraffin-embedded sections were cut at 4 μm, dewaxed in xylene, and heated in a microwave at 60 ℃ for 20 min in EDTA buffer (pH 9.0) for antigen retrieval. For each slide, endogenous peroxidase activity was blocked by a 10 min incubation in 0.3% H2O2 followed by incubation at 37 ℃ with a 1:100 dilution of the primary antibody KSR1. Slides were rinsed three times in PBS, incubated for 30 min with an EnVision staining kit (DAKO), followed by three additional washes in PBS, and color was developed over 3-10 min in a moist chamber at room temperature using 3,3′-diaminobenzidine. Slides were counterstained in hematoxylin and dehydrated in a graded ethyl alcohol series (70%, 90%, and 100%). Assessment of IHC staining was independently performed by two expert pathologists. Any discordance was resolved through discussion and consensus. IHC signal intensities were scored as follows: negative, weak, moderate, or strong.

Supplementary table 1. Correlation analysis of CLTC protein expression in relation to
clinicopathologic variables of 100 patients with osteosarcoma.